Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
  • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54326—Magnetic particles
  • G01N33/5434—Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

• Subject matter of the invention is a reagent preparation for binding components of a sample in the form of a tablet, the use thereof for binding or purifying nucleic acids and a method of preparing a suspension of magnetic particles in a sample, and a method of incorporating magnetic particles in a sample.
• a problem which frequently arises in the analysis of liquid samples is that the components to be analyzed are present only in very minute amounts. Moreover, the sample also contains numerous particles which are not to be determined but render the determination less accurate. It is therefore expedient to bind the analytes to a solid phase and remove the particles which are not to be determined together with the liquid. The isolated analytes can then be detected at the solid phase. Recently, especially the inner walls of reaction vessels such as tubes have been used as solid phases. Another option is to add a bead to the reaction vessel which is capable of binding the analyte. The bead size is such that the separation of liquid and beads can be accomplished by simple pipetting.
• the magnetic particles are provided with a surface capable of binding an analyte.
• Subject matter of the invention is hence a reagent preparation for binding components in a sample in the form of a tablet comprising a multitude of particles having a surface to which the components can essentially completely bind and excipients.
• Another subject matter of the invention is the use of these reagent preparations and a method of preparing magnetic suspensions.
• Components are understood to be particulate or molecular material. This includes especially cells, e.g. viruses or bacteria, but also isolated human or animal cells such as leukocytes, then also immunologically active low and high molecular chemical compounds such as haptens, antigens, antibodies, and nucleic acids. Particularly preferred are nucleic acids such as DNA or RNA.
• Samples as understood in the invention are for example clinical specimen such as blood, serum, mouth wash liquid, urine, cerebrospinal fluid, sputum, stool, punctate, and bone marrow samples.
• the sample can also stem from areas such as environmental analysis, food analysis or molecular-biological research, e.g. bacterial cultures, phage lysates, and products of amplification processes such as PCR.
• a tablet as understood in the invention is a solid, formed body, preferably in the form of a disk or a more or less perfectly shaped sphere. Other similar embodiments are also conceivable. Tablets of this kind are commonly known from drugs.
• a tablet preferably has a defined weight which exceeds 5 mg.
• a magnetic particle is a particle made of a material which can be attracted by a magnet, i.e. ferromagnetic or superparamagnetic materials.
• the invention prefers in particular superparamagnetic particles, especially those that are not premagnetized.
• Premagnetization as understood here is a process of bringing a material into contact with a magnet to increase resonance.
• Magnetide Fe 3 O 4
• Fe 2 O 3 are particularly preferred.
• a magnetic particle is, however, also understood to include materials which contain (smaller) magnetic particles. This includes in particular Iriodin 600 a pigment which is commercially available from Merck (Darmstadt, Germany).
• the invention prefers in particular particles with an average grain size of less than 100 ⁇ m.
• a particularly preferred grain size ranges between 10 and 60 ⁇ m.
• the preferred grain distribution is relatively homogeneous; in particular, there are almost no particles smaller than 10 ⁇ m or larger than 60 ⁇ m. Particles which satisfy this requirement are described for example in WO 90/06045.
• An essential element of the invention is the fact that magnetic particles have a surface to which components can bind. This binding can either be specific or relatively non-specific. Specific binding can be achieved by making use of a binding-specific interactions, e.g. antibodies and antigens, antibodies and haptens or complementary nucleic acids. A combination of these interactions is also possible.
• a known method of modifying a surface is, for example, the coating of particles with a streptavidin layer. It is thus possible to generate a universal matrix to which specific components can be bound from the sample via conjugates of biotin and a certain antibody, hapten or nucleic acid.
• the expert especially one from the field of immunoassays, is familiar with corresponding embodiments.
• a relatively non-specific binding is the interaction between a glass-like surface and nucleic acids.
• the binding of nucleic acids from agarose gel in the presence of sodium iodide in ground flint glass is known from Proc Natl Acad USA 76, 615-619 (1979).
• U.S. Pat. No. 2,233,169 describes magnetic particles whose glass portion can bind nucleic acids.
• the invention proposes that the component to be determined bind essentially completely to the magnetic particles.
• the expert can easily determine the necessary amount of particles by varying the amount of magnetic particles to be added.
• an essentially complete binding means binding of more than 60%, particularly preferred more than 90% of the component to be bound found in the sample.
• Excipients essentially serve to maintain the shape of the tablet, i.e. to link the magnetic particles to form a tablet.
• Preferred excipients of the invention are those which dissolve rapidly in the sample where the reaction is to take place.
• preferred liquid samples are aqueous solutions, it is possible to use those excipients that are usually employed in the manufacture of drugs.
• Polyethyleneglycol (PEG) and polyvinylpyrrolidon (PVP) are particularly preferred.
• DE-A-4406139 describes a magnetic depot drug with improved absorbance of the active components.
• the tablet contains a disk-like magnet and the active component is released over period of several hours.
• the tablet of the invention can also contain stabilizing reagents.
• sugars such as D-mannite, trehalose, and sorbite are added.
• magnetic particles especially those with a glass surface, can be stored in the form of a tablet without visible hydrolysis of the glass and hence without visible elution of the iron from the magnetic portion.
• the magnetic particles are preferably glass magnet pigments or polymer magnetic beads or other magnetic particles with a size ranging between 0.1 ⁇ m and 100 ⁇ m; e.g. those described in DE 19520398.
• the preparation can also contain additives to facilitate the binding process of the components.
• nucleic acids these are chaotropic salts such as guanidinium hydrochloride, sodium iodide, sodium perchlorate or the like. Chaotropic salts of this kind are known from Anal. Biochem. 121, 382-387 (1982) and DE-A 3734442.
• the reagent preparation can also contain reagents which convert the components into a form which basically enables a binding process.
• a reagent is, for example, proteinase K or the above chaotropic salts.
• the reagent preparation can also contain pH buffer substances and reagents for dissolving links such as hydrogen bridges, hydrophobic and ion links as well as reagents for the specific detection of substances or indicators as they are known with components of immunoassays.
• composition has proven to be feasible for a preferred tablet:
• Particularly Preferred Preferred Component Amount Amount Excipient e.g. PEG, PVP, Calcium stearate
• Reagents 0-90% 87% Magnetic particles 0.01-50%
• the tablet of the invention can of course also contain other components, e.g. inert filling agents; the total amount adds up to 100%.
• the percentages given are weight percentages.
• the reagent preparation of the invention in the form of a tablet can be manufactured corresponding to other drugs in tablet form. To accomplish this, all necessary components are thoroughly mixed and aliquots are tabletted in a tablet press. This is accomplished in particular by applying pressure. Tablets of the invention can, however, also be obtained by granulating the mixture of components. For this purpose, a certain amount of the dry mixture is granulated with a solubilizing liquid. Then liquid is again withdrawn from the so obtained granulate. Uniform grain size can be obtained by sieving the granulate.
• the tablets of the invention can be rapidly dissolved, preferably in less than 30 sec., particularly preferred in less than 1 to 10 sec. while the magnetic particles can be easily and readily dispersed. Tablet form is also expedient with respect to storage. Dosing can even be accomplished manually with the aid of a tablet dispenser. Adulterations which occur in suspensions and are caused by sedimentation of particles have not been observed.
• Another subject matter of the invention is the use of the reagent preparation for binding nucleic acids.
• the reagent preparation is added to the sample and incubate until (1.) the tablet has dissolved and (2.) the nucleic acids are essentially completely bound to the surface.
• the tablet can be mechanically moved, if necessary. This increases both the dissolving rate of the tablet and the binding rate of the components.
• Another subject matter of the invention is the use of the reagent preparation for purifying nucleic acids.
• the magnetic particles and the nucleic acids bound thereto are separated from the surrounding sample liquid. This is advantageously accomplished in that a magnetic field is applied to retain the magnetic particles in a vessel or at a defined site of the apparatus; then the sample liquid is removed (by e.g. pipetting or displacement) and, if desired, one or several washing steps with other liquids are performed. If desired, the bound nucleic acids can be separated again from the magnetic particles when suitable conditions are applied. In the case of a glass-like surface, these are low-salt conditions, i.e. the salt contents of the elution solution is less than 100 mmol/l.
• Another subject matter of the invention is a method of preparing a suspension magnetic particles in a sample comprising the steps of adding to the sample a tablet containing magnetic particles and soluble excipients and moving the tablet in sample, preferably with the aid of a movable magnetic field.
• the magnetic field can be moved in that a magnet in the vicinity of the sample is moved back and forth such that the magnetic particles are subject to continuous movement. It is, however, also possible that the vessel containing the sample with the tablet and the magnetic particles is moved with respect to the magnet.
• a two-phase mixture is obtained which is stirred at room temperature until one single phase is obtained. Then 37.8 ml trimethylborate are added dropwise. Subsequently the sol is for 2 hours kept at a temperature of 50° C. Then, 14.1 ml of 15 M HCl are added.
• Iriodin 600 Black Mica, Merck, Darmstadt, Germany
• a spray-drier Bücchi 190, Mini Spray Dryer
• the powder obtained in the spray-drying process was then subject to temperature treatment under a nitrogen atmosphere.
• the heating rate was 1 K/min and the dwelling time was 2 hours at the compacting temperature.
• the temperature was lowered down to the temperature of the follow-up treatment; the nitrogen atmosphere was replaced by air and after the follow-up treatment, the powder was cooled down to room temperature.
• Agglomerates that may have formed were removed by sieving with a 50 ⁇ m sieve.
• Tris-HCl and 0.259 g urea were dissolved in 2.2 ml bidest. water. Together with 266.4 g of the pre-mix, the solution was then grained. A total of 7 ml H 2 O bidest were added. The resulting granulate was dried in a vacuum at room temperature over a period of 24 hours and subsequently sieved through a 0.6 mm sieve.
• Example 2 10 tablets of Example 2 were weighed and added into an open glass vessel and stored open in the lab at room temperature at appr. 50% rF. The weight was monitored over a period of four weeks. There were no weight changes.
• the magnetic particles were washed by mixing for 10 seconds, incubating for 1 min at RT, and mixing again for 10 seconds, and then deposited at the vessel wall using the magnetic particle separator. The supernatant was removed and discarded. The washing procedure was repeated until the washing supernatant was colorless (total of 5 times) Now, the nucleic acids were eluted by mixing 3 times for 10 seconds using 200 ⁇ l each time and elution buffer that was preheated to 70° C.; then incubated again at for 10 min. at RT and mixed again for 10 min.
• the supernatant obtained after the first binding to the magnetic glass particle was checked for its contents of nucleic acids as follows: The supernatant was transferred into a filter tube (Boehringer Mannheim Cat. no. 1744003, contained in the High Pure PCR Product Purification Kit) and centrifuged for 1 min at 8000 rpm using an Eppendorf table centrifuge). The flow-through was discarded and the filter tube washed 2 ⁇ with 500 ⁇ l washing buffer each time (centrifuged again as before). The filter tube was centrifuged until it was dry and then eluted by repeating the centrifugation and using 2 ⁇ 200 ⁇ l 1 ⁇ eluting buffer preheated to 70° C.
• a filter tube Boehringer Mannheim Cat. no. 1744003, contained in the High Pure PCR Product Purification Kit
• sample buffer 10 ⁇ l were added to 50 ⁇ l of the eluate and the supernatant processed via the filter tube; 45 ⁇ l thereof were then for 90 minutes electrophoretically separated at 120 V in a 0.8% agarose gel.
• sample buffer 10 ⁇ l sample buffer were added to the 50 ⁇ l PCR batches and 45 ⁇ l thereof were then for 90 minutes electrophoretically separated at 120 V in a 0.8% agarose gel.
• example 2 tablets of example 2 were added into an Eppendorf reaction vessel, 40 nl proteinase K (20 mg/ml) and 200 ⁇ l thawed whole blood (cf. example 4a) were added and immediately mixed for 10 seconds on a vortex mixer. 200 ⁇ l of 30% Triton X-100 were added and mixed for 10 min on the vortex mixer. The treatment was then continued as described in example 4.

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• Bioinformatics & Cheminformatics (AREA)
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• General Engineering & Computer Science (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Medicinal Preparation (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Apparatus Associated With Microorganisms And Enzymes (AREA)
• Sampling And Sample Adjustment (AREA)

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Citations (18)

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• 1996
  • 1996-06-07 DE DE19622885A patent/DE19622885A1/de not_active Withdrawn
• 1997
  • 1997-05-29 AU AU23705/97A patent/AU715803B2/en not_active Ceased
  • 1997-06-04 CA CA002206760A patent/CA2206760C/en not_active Expired - Lifetime
  • 1997-06-05 JP JP16504297A patent/JP3821541B2/ja not_active Expired - Lifetime
  • 1997-06-05 IL IL12100897A patent/IL121008A/en not_active IP Right Cessation
  • 1997-06-06 DK DK97109177T patent/DK0811694T3/da active
  • 1997-06-06 DE DE59709467T patent/DE59709467D1/de not_active Expired - Lifetime
  • 1997-06-06 EP EP97109177A patent/EP0811694B1/de not_active Expired - Lifetime
  • 1997-06-06 US US08/870,385 patent/US6274386B1/en not_active Expired - Lifetime
  • 1997-06-06 ES ES97109177T patent/ES2195053T3/es not_active Expired - Lifetime
  • 1997-06-06 AT AT97109177T patent/ATE234366T1/de active
  • 1997-06-07 KR KR1019970023487A patent/KR100260326B1/ko not_active IP Right Cessation
• 2001
  • 2001-06-29 US US09/893,533 patent/US6746874B2/en not_active Expired - Lifetime

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